Human infections caused by Staphylococcus aureus present a serious therapeutic challenge due to the appearance of antibiotic-resistant strains. The mechanism of surface protein anchoring to the S. aureus cell wall is examined as a possible target for anti-infective therapy. Staphylococcal surface proteins are synthesized as precursors with an N-terminal signal peptide and a C-terminal sorting signal, containing a LPXTG motif as well as positively charged residues. The charged residues are thought to retain surface proteins within the secretion pathway, allowing sorting signal cleavage between the threonine (T) and the glycine (G) of the LPXTG motif. The carboxyl-group of threonine is subsequently amide-linked to the amino-group of peptidoglycan crossbridges, anchoring the C-terminal ends of surface proteins to the staphylococcal cell wall. Sortase (SrtA), a membrane anchored transpeptidase of S. aureus, is responsible for anchoring surface proteins with a LPXTG motif to the cell wall envelope. We report here the identification of a second sortase (SrtB) in the Gram-positive pathogen Staphylococcus aureus that is required for anchoring of a hitherto unknown surface protein with a NPQTN motif. Purified SrtB cleaves NPQTN-bearing peptides in vitro, and a srtB mutant is defective in the persistence of animal infections. SrtB is part of an iron-regulated locus called iron-responsive surface determinants (isd), which also contains a ferrichrome transporter and surface proteins with NPQTN and LPXTG motifs. Thus, cell wall anchored surface proteins and the isd locus appear to be involved in a novel mechanism of iron acquisition that is important for bacterial pathogenesis. This proposal aims to characterize the role of sortase B in anchoring surface proteins to the cell wall envelope and to study the contribution of SrtB-mediated cell wall sorting during infection. Genetic and biochemical experiments aim at the identification of genes or gene products that are required for the anchoring of NPQTN-sorting signal containing surface proteins to the cell wall envelope. Other experiments examine the physiological role of the isd locus in iron transport.